Enterococci cause about 5.15% of cases of infective endocarditis an are now the second most common pathogens isolated from hospital acquired infections. The presence of multiple antimicrobial resistances may be partly responsible for this latter role and certainly make it more difficult to successfully treat patients when they become infected with enterococci. The therapeutic dilemmas posed by enterococci have drastically worsened over the past decade with the emergence of high- level resistance to all aminoglycosides, beta-lactamase and non-beta- lactamase mediated resistance to penicillins, and resistance to vancomycin, the most recent and most devastating of all these traits. Because of our rapidly declining antimicrobial options, it seems appropriate and timely to increase investigative efforts to delineate pathogenic mechanisms of enterococci. It is hoped that by understanding these mechanisms, other therapeutic or preventative modalities can eventually be developed, and, indeed, it is now critical that other modalities be pursued. The basic plan of this proposal is to develop the genetic methods to generate knock-out mutations in enterococcal genes and then to use this methodology to inactivate enterococcal genes encoding immunoreactive as well as secreted and surface protein antigens in an enterococcal host. To achieve this goal, we plan to engineer a Tn5 derivative containing an antibiotic resistance gene selectable in both gram-positive and gram-negative hosts. To develop the methodology and demonstrate the utility of this transposon, we will use a series of enterococcal clones which we have generated that complement E. coli auxotrophic mutants. The Tn5 derivative will be used to inactivate the complementing function in E. coli, and then the inactivated clone will be introduced into enterococci, selecting for expression of the antibiotic resistance marker. Heritable resistance is due to incorporation of the inactivated gene into the bacterial chromosome by recombination and should be accompanied by the appearance of a new growth requirement for the enterococcal host, demonstrated on our defined medium. We then plan to use serum from rabbits immunized against enterococcal surface and secreted proteins as well as serum from patients with enterococcal infections that have demonstrable antienterococcal antibodies to identify immunoreactive clones produced by enterococcal DNA libraries cloned into E. coli. This will be done with a cosmid vector which can be mobilized back into enterococci by conjugation or, if needed, an expression vector. The transposon will then be used to isolate insertions in the E. coli clones and mutants that have lost immunoreactivity will be selected. These will be introduced back into enterococci which should result in the insertion of the antibiotic resistance gene into the gene encoding the immunoreactive protein. This in turn should result in inactivation of the wild type enterococcal gene. Enterococci that are isogenic except for specific genes will then be compared in an animal model of peritonitis and in a PMN killing assay to investigate the biologic consequence of inactivation of these genes. The techniques developed during this project should have wide applicability to studies of other genes. The techniques developed during this project should have wide applicability to studies of other genes in enterococi and other gram-positive organisms.